Na(+)-K(+)-ATPase in endothelial cell energetics: 23Na nuclear magnetic resonance and calorimetry study

Abstract

Sodium flux rate and energy consumption of the Na(+)-K+ pump in vascular endothelial cells of porcine aorta grown on micro-carrier beads were studied using a combination of nuclear magnetic resonance spectroscopy of intracellular 23Na and microcalorimetry. The Na+ flux into the cells was determined in the presence of the shift reagent Dy(P3O10)2(7-), while the Na(+)-K+ pump was inhibited with ouabain. Basal Na+ influx was 17 +/- 3 nmol.min-1.mg protein-1, and intracellular Na+ concentration was 23.5 +/- 3.8 mM, resulting in a complete exchange of intracellular Na+ within 5–6 min. Spin-lattice relaxation time (T1) measurements of intracellular Na+ showed a T1 of 19 +/- 1 ms under basal conditions and a T1 of 26.2 +/- 1.6 ms after pump inhibition with 50 microM ouabain. Such an increase is typical for a system in which the total amount of Na+ increases but where the amount of bound Na+ remains constant. The Na+ ionophore nystatin maximally increased the Na(+)-K+ pump rate about twofold, whereas the amount of intracellular Na+ only increased 14%. With microcalorimetry a cellular heat flux of 183 +/- 18 microW per mg endothelial protein was determined, which relates to 7.6 microW/mg endothelial protein generated by the Na(+)-K(+)-adenosinetriphosphatase. Our data demonstrate that small intracellular changes of Na+ can stimulate the endothelial Na(+)-K+ pump activity. The contribution of the Na(+)-K+ pump to total endothelial energy expenditure is approximately 4-5%.